The invention relates to several fields, especially to the detection of specific nucleic acids, proteins, carbohydrates, or organic compounds immobilized on a solid surface. More particularly it relates to detection systems in which the immobilized target is recognized by a metallic nanoparticle probe and for which the signal for detection has been amplified by reductive deposition of silver on the nanoparticle probe.
(a) Gold Nanoparticle Probes
The use of gold nanoparticle probes as reporter for detection of biological polymers was first described by W. P. Faulk and G. M. Taylor, who employed the nanoparticles as immunocytochemical probes for surface antigens [Immunochemitry, 8, 1081 (1971)]. Since then gold colloids have been widely used for detection of a variety of proteins using electron or light-microscopy to observe the particles [for reviews see Hacker, G. W. in Colloidal Gold; Principles, Methods, and Applications, Vol. 1, Academic Press, Inc. (1998) p 297, and Garzon, S., and Bendayan, M. in Immuno-Gold Electron Microscopy in Virus Diagnosis and Research, Ed. Hyatt, A. D. and Eaton, B. T., CRC Press, Ann Arbor, (1993) p 137]. Recently, applications of gold nanoparticle or cluster conjugates as probes for detection of oligonucleotides and nucleic acids have been suggested [Kidwell, D. A., and Conyers, S. M., U.S. Pat. No. 5,384,265 (1995); Hainfeld, J. F., et al. U.S. Pat. No. 5,521,289 (1996)] and described [Tomlinson, S., et al., Analytical Biochemistry, 171, 217 (1988); Mirkin et al., Nature, 15, 607 (1996); Storhoff, J. J. et al., J. Am. Chem. Soc., 120, 1959 (1998)].
(b) Silver Enhancement of Signal
It has been found that the sensitivity for assays utilizing gold markers for proteins in tissues [Danscher, G. Histochemistry, 71, 1 (1981); Holgate, C. S. et al. J. Histochem. Cytochem. 31, 938 (1983)], for nucleic acids visualized in situ in immobilized biological systems [Gassell, G. J., et al., J. Cell Biology, 126, 863 (1994); Zehbe, I. et al., Am J. of Pathology, 150, 1553 (1997); Hacker, G. W., Eur. J. Histochem 42, 111 (1998) and for gold probes targeted to oligonucleotides captured on oligonucleotide arrays on a glass surface [T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science, 289, 1757 (2000)] can be significantly increased by silver staining. In this process, the gold particles captured on a surface are treated with a solution containing silver ions and a reducing agent (e.g., hydroquinone). The gold catalyzes reduction of the silver ions so that silver is deposited on the gold particle, and the early-deposited silver can itself catalyze further reduction of silver ion. As a consequence, the amount of metal that can be visualized is greatly increased. Unfortunately, however, the silver reduction catalyzed by the deposited silver ceases after a time, so the extent of amplification achievable is limited. When employed in enhancing visibility of gold nanoparticles on a glass plate, one observes darkening of the spot characteristic for the gold probes captured by a target sequence. Indeed, a good silver spot may be observed for cases where the amount of gold deposited initially is too small to be visible to the naked eye. Typically, the reaction time for the silver staining step is short, of the order of five minutes or less. Long exposure to the silver solution leads to non-selective deposition of silver metal and a high background. The silver ion solution and the reducing agent are mixed just prior to application to minimize the uncatalyzed reduction.
(c) Oligo- and Polynucleotide Arrays
A recent major innovation in biology utilizes arrays of oligonucleotides or polynucleotides tethered to a solid surface. These oligomers serve as capture probes to bind complementary DNA or RNA target sequences. The captured sequences can in turn be recognized by fluorescent labels previously attached to them or by fluorescent or calorimetric probes that bind to a segment of the target. As stated by Eric Lander [Nature Genetics Supplement, 21, 3 (1999)]: xe2x80x9cArrays offer the first great hope . . . by providing a systematic way to survey DNA and RNA variation. They seem likely to become a standard tool of both molecular biology research and clinical diagnostics. These prospects have attracted great interest and investment from both the public and private sectors.xe2x80x9d
Array technology is indeed now greatly accelerating developments in our understanding of genetic variation and gene expression. Nevertheless, current methodology suffers from several limitations, an important one being relatively low sensitivity in detecting fluorescently labeled targets on the chip arrays. Typically, targets in the range of picomolar concentrations or higher must be employed. Genetic analyses of natural targets in the attomolar or zeptomolar range therefore require target amplification by PCR. This procedure demands time and labor, and the target amplification can lead to errors in the sequence to be tested.
A need exits for a more sensitive, simpler, and cheaper detection method for polynucleotides arrayed on chips. Progress in detection technology has been made with the use of gold nanoparticle oligonucleotide conjugates as probes and signal amplification by silver ion reduction, which enables assays of polynucleotides of 50 fM concentration to be readily detected [for the methodology, see T. A. Taton, C. A. Mirkin; R. L. Letsinger, Science, 289, 1757 (2000). We describe here a discovery that significantly lowers further the target concentration required for assays employing gold nanoparticles and other metallic nanoparticles.
The present invention relates to a method for amplifying signal by enhancing the deposition of silver in detecting systems where the formation of a silver spot serves as a reporter for the presence of a molecule, including biological polymers (e.g., proteins and nucleic acids) and small molecules. The detecting systems include detection of molecules in situ (e.g., on cells or in a tissue sample) and assays where the molecule to be detected (the target molecule) is bound to a substrate or is captured by another molecule bound to a substrate (the capture molecule). The invention has special utility in increasing the signal strength in diagnostic and screening applications involving detection of target molecules arrayed at discrete positions on a solid surface. It, therefore, provides a means for greatly enhancing the sensitivity of tests carried out on microarrays or microchips. The process is distinguished by the simplicity and economy of its execution and the large enhancement in signal and, thereby, sensitivity realized.
This invention is based on the discoveries that (1) gold nanoparticles coated with oligonucleotides bind to silver that had previously been deposited on gold nanoparticle-oligonucleotide conjugates immobilized by hybridization on a glass substrate or plate and (2) that the resulting (gold nanoparticle-oligonucleotide-silver-(gold-oligonucleotide) structures function as catalyst for the further deposition of silver by reduction of silver ions. The first discovery is surprising since one might expect that the surface bound oligonucleotides, which shield the nanoparticles from non-specific binding to the glass surface and the oligonucleotides immobilized on the glass surface, would also shield the nanoparticles against interaction with the silver surface. Indeed, other work has shown that oligonucleotides protect gold nanoparticle oligonucleotide conjugates from fusing to form gold-gold bonds between individual nanoparticles even when the mixtures are dried. The second discovery is significant since it provides a means for substantially increasing the metallic mass at the site of the originally immobilized nanoparticles. In conjunction with development of buffer conditions that enable oligonucleotide nanoparticle conjugates that are unbound by hybridization or interaction with silver to be washed cleanly from the glass surface these findings opened opportunities for assaying polynucleotide targets at extremely low target concentrations.
Accordingly, one objective of the invention is to provide a method for amplifying a detection signal comprising:
(a) providing a substrate having deposited silver;
(b) contacting the substrate having deposited silver with a solution comprising nanoparticles having oligonucleotides bound thereto so as to produce a substrate having a nanoparticles-silver sandwich;
(c) washing the substrate having said sandwich; and
(d) contacting the substrate having said sandwich with silver ions and a reducing agent to promote silver deposition onto the nanoparticles of said sandwich.
The nanoparticles having oligonucleotides bound thereto comprise gold, silver, platinum or mixtures thereof. These nanoparticles may be in the form of gold nanoparticle-oligonucleotide conjugates or complexes.
Another object of the invention is to provide a method for promoting silver deposition onto a surface comprising silver, said method comprising the steps of:
(a) providing a surface having silver bound thereto;
(b) contacting the surface with a solution comprising nanoparticles having oligonucleotides bound thereto so as to produce a surface having a nanoparticles-silver sandwich;
(c) washing the surface having said nanoparticles-silver sandwich;
(d) contacting the surface having said nanoparticles-silver sandwich with a solution including silver ions under reducing conditions to promote silver deposition onto said nanoparticles of said nanoparticles-silver sandwich; and
(e) washing the surface having deposited silver.
According to this method, the surface may include cells or tissue for in situ detection of target molecules. Preferrably the nanoparticles having oligonucleotides bound thereto comprise gold nanoparticles having oligonucleotides bound thereto in conjugate or complex form. In practicing this invention, gold nanoparticle oligonucleotide conjugates are preferred.
A further object of the invention is to provide a kit for detection signal amplification comprising:
(a) container including nanoparticles having oligonucleotides bound thereto;
(b) container including a silver salt; and
(c) container including a reducing agent.
The kit may include instructions for use in amplifying silver stain detection signals. Preferrably the nanoparticles having oligonucleotides bound thereto comprise gold nanoparticles having oligonucleotides bound thereto in conjugate or complex form. In practicing this invention, gold nanoparticle oligonucleotide conjugates are preferred.